1. Field of the Invention
This invention relates to a novel colorimetric method for beta-lactamase assay and its applications.
2. Description of the Prior Art
The amide bond in a beta-lactam ring of a beta-lactam antibiotic can be hydrolyzed enzymatically by beta-lactamases or non-enzymatically in neutral or alkaline aqueous solution to form an open beta-lactam ring end product. The open beta-lactam ring end product resulting from the hydrolysis of a penicillin, catalyzed by beta-lactamase, is the corresponding penicilloic acid, which is stable; while that resulting from the hydrolysis of a cephalosporin is the corresponding cephalosporoic acid, which is often unstable [Ross G. W., and C. H. O'Callaghan, Methods in Enzymology 43: 69-85 (1975)]. When the beta-lactam ring of a penicillin is hydrolyzed non-enzymatically, the corresponding penicilloic acid is a mixture of its diastereomers [Levine, B. B., Nature 187: 939-940 (1960)]. The iodometric method is one of the colorimetric methods developed for qualitative or quantitative assay of beta-lactamase. It is based on the decolorization of a chromophore by the open beta-lactam ring end product, but not by its intact beta-lactam substrate. A microiodometric method which employs a starch-iodine complex as the chromophore is widely used for beta-lactamase assay [Novick, R. P., Biochem. J. 83: 236-240 ( 1962); Sykes, R. B., and K. Nordstrom, Antimicrob. Agents Chemother. 1: 94-99 (1972)]. However, since the iodine molecules in the iodine-starch complex sometimes inactivate the enzyme, the microiodometric method cannot be used universally for beta-lactamase assay. The newly developed fluorescence assay methods and several other methods for detection of microbial beta-lactamases have been described in U.S. Pat. Nos. 4,740,459 (1988) to Chen, K. C. S., J. Knapp and K. K. Holmes; and 4,965,193 (1990) to Chen, K. C. S. Although the fluorescence assay methods disclosed in the previous patents are sensitive and specific for detecting microbial beta-lactamases, they all require a long-wave ultraviolet lamp and a dark chamber for viewing the fluorescent end products.
The cytochrome c oxidase test (known as the oxidase test) is a procedure for detecting the presence of the cytochrome c oxidase system in bacteria, and is important in identifying and/or differentiating certain microorganisms. In the oxidase test, cytochrome c oxidase catalyzes the oxidation of reduced cytochrome c by molecular oxygen. The test reagent, an N-alkyl derivative of p-phenylenediamine such as N,N-dimethyl-p-phenylenediamine (DMP), or N,N,N',N'-tetramethyl-p-phenylenediamine (TMP), is then oxidized to a colored compound by the oxidized cytochrome c, which in turn changes to reduced cytochrome c [Lanyi, B., Methods in Microbiology 19: 1-67 (1987)]. Some oxidase-positive microorganisms also produce penicillinases. Current methods require two separate tests for detecting the microbial cytochrome c oxidase system and penicillinase.
Gonorrhea remains one of the most widespread sexually transmitted diseases in the world. Since strains of the penicillinase-producing Neisseria gonorrhoeae (PPNG) were first isolated in 1976 [Phillips, I., Lancet ii: 656-657 (1976); Ashford, W. A. et al., Lancet ii: 657-658 (1976)], gonococcal infections caused by PPNG have increased dramatically in the United States and other parts of the world. In 1986, PPNG infections accounted for approximately 2% of gonorrhea reported in the United States [Morbid. Mortal. Weekly Rep. 36: 107-108 (1987)] and in 1988 and 1989, PPNG accounted for 3.2% and 7.4% of the isolates, respectively, at 21 selected public clinics in 21 major U.S. cities [Morbid. Mortal. Weekly Rep. 39: 284-287 (1990)]. In Southeast Asia, the isolation rates of PPNG strains have remained high. The incidences in several countries were recorded as follows: Japan 16.1%; Malaysia 37.2%; Thailand 42%; South Korea 40+%; the Philippines 30-40%; Indonesia 25%; and Singapore 25% [Sng, E. H. et al., Br. J. Vener. Dis. 60: 374-379 (1984)]. With the exception of allergic reactions, penicillin antibiotics are free of adverse side effects and relatively inexpensive for treating gonorrhea, but they cannot be used to eradicate PPNG. Instead, more expensive drugs which often require parenteral administration and cannot eradicate coexisting syphilis, are employed [Kraus, S. J. et al., Sex. Transm. Dis. 15: 234-243 (1988)].
Gonorrhea is diagnosed by the recognition of typical Gram-negative intracellular diplococci in stained smears and by identification of N. gonorrhoeae in cultures of secretions. In a clinical laboratory, identification of N. gonorrhoeae in urethral exudates requires culture of the organism, which takes approximately 24-72 hours. Differentiation of the infecting gonococci into PPNG and non-PPNG requires further testing for penicillinase production after culture. Because of the time necessary for analysis, the outcome of the laboratory testing is rarely of use to the physician in initial therapy. Therefore, most patients are treated empirically on the basis of a Gram stain. The oxidase test has long been used for identifying N. gonorrhoeae [Steel K. J., J. Gen. Microbiol. 25: 297-306 (1961)]. U.S. Pat. Nos. 3,876,503 (1975), 3,954,563 (1976), 3,954,564 (1976), 4,018,653 (1977), 4,108,729 (1978), 4,340,670 (1982) and 4,355,113 (1982), all issued to F. C. Mennen, disclose several devices containing TMP or DMP as the oxidase reagent for presumptive identification of N. gonorrhoeae in urethral exudates. A three-minute nonculture method for presumptive identification of N. gonorrhoeae in urethral exudates from men with urethral discharge was reported [Janda W. M., and T. Jackson, J. Clin. Microbiol. 21: 143-145 (1985)]. This method used a device containing TMP as the oxidase reagent to detect the cytochrome c oxidase systems (presumably those of the infecting gonococci in urethral exudates). This test had a sensitivity of 95.6% and a specificity of 84.2% for identification of N. gonorrhoeae in urethral exudates compared with the culture method; and the same results showed no significant difference compared with the Gram stain method. In a community, if the incidence of PPNG infection is high, the routine use of penicillin cannot be recommended for primary therapy unless penicillinase production in urethral specimens can first be excluded by a rapid screen test. A fluorescent spot test method [Chen, K. C. S., and K. K. Holmes, J. Clin. Microbiol. 23: 539-544 (1986)] was applied to the rapid and direct detection of PPNG in male urethral exudates [ Taylor, D. N. et al., Lancet ii: 625-626 (1985)]. This method requires a long-wave ultraviolet lamp and a dark chamber for viewing the fluorescent end products. There are no methods for simultaneously detecting cytochrome c oxidase and penicillinase in urethral exudates for presumptive identification of N. gonorrhoeae and differentiation of the infecting gonococci into PPNG and non-PPNG.
Enzyme labels are required for an enzyme immunoassay (EIA) and a non-radioisotope DNA probe. The enzymes commonly used as labels are: horseradish peroxidase (HRP), glucose oxidase, alkaline phosphatase, beta-galactosidase, and urease. Recently, penicillinase was also used as an enzyme label for EIA [Yolken, R. H. et al., J. Immunol. Methods 73: 109-123 (1984)]. A simultaneous colorimetric assay method for any pair of the enzymes described above is currently not available, because the color signals produced by the two enzymes interfere with each other. Therefore, using current EIA or non-radioisotope DNA probe methods, two analytes in a specimen cannot be determined simultaneously.
This invention discloses a novel colorimetric assay for the open beta-lactam ring end product resulting from the hydrolysis of a beta-lactam substrate. It is an object of the present invention to provide a specific colorimetric assay for beta-lactamase including detection of microbial beta-lactamases. The chromophores used for beta-lactamase assay do not inactivate the beta-lactamase; therefore the assay method disclosed in this invention can be generally applied.
It is also an object of the present invention to provide a method for simultaneous detection of microbial cytochrome c oxidase systems and penicillinases to save time and decrease the number of organisms needed from culture for performing these two biochemical tests.
Further, it is an object of the present invention to provide a rapid nonculture method for simultaneous presumptive identification of N. gonorrhoeae in urethral exudates and differentiation of the infecting gonococci into PPNG and non-PPNG to facilitate the initial treatment for gonorrhea.
Another object of the present invention is to provide a colorimetric method for simultaneous assay for peroxidase and penicillinase using the same wavelength. Therefore, it provides great potential for analyzing two analytes in an incubation mixture using methods of EIA and non-radioisotope DNA probe with double enzyme labels.
Further objects and advantages will become apparent from consideration of the ensuing examples.